Fungal multienzyme production on industrial by-products of the citrus-processing industry

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 2373–2383

Fungal multienzyme production on industrial by-products of the citrus-processing industry Diomi Mamma, Elisavet Kourtoglou, Paul Christakopoulos

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Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 157 80 Zografou, Attica, Greece Received 26 January 2007; received in revised form 16 March 2007; accepted 5 May 2007 Available online 28 June 2007

Abstract Orange peels is the principal solid by-product of the citrus processing industry and the disposal of the fresh peels is becoming a major problem to many factories. Dry citrus peels are rich in pectin, cellulose and hemicellulose and may be used as a fermentation substrate. Production of multienzyme preparations containing pectinolytic, cellulolytic and xylanolytic enzymes by the mesophilic fungi Aspergillus niger BTL, Fusarium oxysporum F3, Neurospora crassa DSM 1129 and Penicillium decumbens under solid-state fermentation (SSF) on dry orange peels was enhanced by optimization of initial pH of the culture medium and initial moisture level. Under optimal conditions A. niger BTL was by far the most potent strain in polygalacturonase and pectate lyase, production followed by F. oxysporum F3, N. crassa DSM 1129 and P. decumbens. N. crassa DSM 1129 produced the highest endoglucanase activity and P. decumbens the lowest one. Comparison of xylanase production revealed that A. niger BTL produced the highest activity followed by N. crassa DSM 1129, P. decumbens and F. oxysporum F3. N. crassa DSM 1129 and P. decumbens did not produce any b-xylosidase activity, while A. niger BTL produced approximately 10 times more b-xylosidase than F. oxysporum F3. The highest invertase activity was produced by A. niger BTL while the lowest ones by F. oxysporum F3 and P. decumbens. After SSF of the four fungi, under optimal conditions, the fermented substrate was either directly exposed to autohydrolysis or new material was added, and the in situ produced multienzyme systems were successfully used for the partial degradation of orange peels polysaccharides and the liberation of fermentable sugars.  2007 Elsevier Ltd. All rights reserved. Keywords: Orange peels; Multienzyme production; Hydrolysis

1. Introduction Citrus fruits constitute an important group of fruit crops produced all over the world. The family of citrus fruits consists of Oranges, Kinnow, Khatta, Lime, Lemon, Grapefruit, Malta, Sweet orange etc. World citrus production has increased significantly since the 1980s. For example, in 2010 the orange production is estimated to reach 66.4 million MT, which represents a 14% increase compared to that of 1997–1999. About 30.1 million MT of the orange production will be processed to yield juice, essential oils

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Corresponding author. E-mail address: [email protected] (P. Christakopoulos).

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.05.018

and other by-products (FAO, 2003). Citrus peels is the principal solid by-product of the citrus processing industry and constitutes about 50% of fresh fruit weight (Garzo´n and Hours, 1992). The disposal of the fresh peels is becoming a major problem to many factories. Usually, citrus juice industries dry the residue and it is either sold as raw material for pectin extraction or pelletized for animal feeding, though none of these processes is very profitable (Garzo´n and Hours, 1992). In Greece approximately, 35,000 tons (dry weight) of citrus peels are produced each year, and only a small fraction is used as cattle food (Ververis et al., 2007). Various microbial transformations have been proposed for the utilization of food processing waste for producing valuable products like biogas, ethanol, citric acid, chemicals, various enzymes, volatile flavouring

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compounds, fatty acids and microbial biomass (Dhillon et al., 2004). Dry citrus peels are rich in pectin, cellulose and hemicellulose (Ismail, 1996). Pectins are a family of complex polysaccharides that contain 1,4-linked a-D-galactosyluronic acid (GalpA) residues. Three pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I, and substituted galacturonans) have been isolated from primary cell walls and structurally characterized (Ridley et al., 2001). Pectinolytic enzymes or pectinases are a heterogeneous group of related enzymes that hydrolyze the pectic substances. An extensive classification of pectinolytic enzymes was made by Jayani et al. (2005). Pectinolytic enzymes are of significant importance in the current biotechnological era with their all embracing applications in fruit juice extraction and its clarification, scouring of cotton, degumming of plant fibers, waste water treatment, vegetable oil extraction, tea and coffee fermentations, bleaching of paper, in poultry feed additives and in the alcoholic beverages and food industries (Jayani et al., 2005). Xylan is the principal component of plant cell wall hemicelluloses. It is a heteroglycan composed of a linear chain of xylopyranose residues bound by b(1 ! 4) linkages, with a variety of substituents linked to the main chain by glycosidic or ester linkages. The xylanolytic enzyme system carrying out the xylan hydrolysis is usually composed of a repertoire of hydrolytic enzymes: b-1,4-endoxylanase, b-xylosidase, a-L-arabinofuranosidase, a-glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase. Xylanolytic enzymes have attracted a great deal of attention in the last decade, particularly because of their biotechnological potential in various industrial processes such as food, feed, and pulp and paper industries (Beg et al., 2001). Cellulose consists of a simple chemical structure (b-1,4 linked glucose homopolymer). Cellulases are a complex enzyme system, comprising endo-1,4-b-D-glucanase, exo1,4-b-glucanase and b-D-glucosidase. These enzymes are employed in feed, fuel and chemical industries for the processing of lignocellulosic materials (Pandey et al., 1999). Among processes used for enzyme production, solidstate fermentation (SSF), which can be defined as ‘‘the growth of microorganisms (mainly fungi) on moist solid materials in the absence of free-flowing water’’ (PerezGuerra et al., 2003), is an attractive one because it presents higher productivity per reactor volume, lower capital and operating costs, lower space requirements, simpler equipment and easier downstream processing compared to that of submerged fermentation (SmF) (Pandey et al., 2000a). Several authors have reviewed the different applications of solid-state fermentation (Raimbault, 1998; Pandey et al., 2000b; Perez-Guerra et al., 2003; Ho¨lker and Lenz, 2005). There is also evidence that some enzymes are less affected by catabolic repression, than those obtained by SmF (Martins et al., 2002).

The use of SSF for pectinase production has been proposed using different solid agricultural and agro-industrial residues as substrates such as wheat bran (Castilho et al., 1999; Soares et al., 1999; Singh et al., 1999), soy bran (Castilho et al., 2000), apple pomace (Hours et al., 1988; Hang and Woodanms, 1994), cranberry and strawberry pomace (Zheng and Shetty, 2000), beet pulp (Spagnuolo et al., 1997; Sidi et al., 1984), coffee pulp and husk (Boccas et al., 1994; Antier et al., 1993), cocoa (Schwan et al., 1997), lemon and orange peel (Garzo´n and Hours, 1992; Fonseca and Said, 1994; Larios et al., 1989; Maldonado et al., 1986) and sugar cane bagasse (Acun˜a-Argu¨elles et al., 1994). Some of the substrates that have been used for the production of cellulolytic and hemicellulolytic enzymes under SSF included wheat bran, wheat straw, rice bran, rice straw, rice husk, corncobs, corn straw, sugar beet pulp, sago hampas, coconut coir pith, steam pre-treated willow, grapevine trimmings dust, apple pomace, cassava waste, tea waste, soyhull, palm oil mill waste, sweet sorghum pulp, aspen pulp (Pandey et al., 1999). Wheat bran however holds the key, and has most commonly been used, in various processes. The present work aims at the utilization of orange peels through its microbial biodegradation by fungal strains of the genera Aspergillus, Fusarium, Neurospora and Penicillium, for the production of multienzyme preparations containing, pectinases (polygalacturonase EC 3.2.1.15, pectate lyase EC 4.2.2.2), cellulases (endoglucanase EC 3.2.1.4 and b-glucosidase EC 3.2.1.21) xylanases (endoxylanase EC 3.2.1.8 and b-xylosidase EC 3.2.1.37) and invertase (EC 3.2.1.26). Furthermore, owing to the high cost of pure enzymes, the use of a simple two-stage process is proposed which includes the SSF growth of the four fungi on orange peels followed by the hydrolysis either of the fermented substrate (autohydrolysis) or the added amounts of it and the conversion of polymeric carbohydrates to monomeric sugars for further conversion to value-added products. 2. Methods 2.1. Microorganisms The microorganisms used throughout the present study were: (a) the wild-type strain F3 of Fusarium oxysporum, isolated from cumin (Christakopoulos et al., 1989), (b) the wild-type strain of Aspergillus niger (strain BTL) isolated from corn (Christakopoulos et al., 1990), (c) Neurospora crassa DSM 1129 which was obtained from DSMZ (Deutsche Sammlung von Microorganismen und Zellkulture GmbH), Germany and (d) the wild-type strain of Penicillium decumbens which was kindly provided by Professor Li Ze-Lin, Shichuan Academy of Food and Fermentation Industries, Chengdu, PR China. Stock cultures were maintained on potato dextrose agar (PDA) slants at 4 C.

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2.2. Materials Dry orange peels were provided by a local orange processing industry. Dry material was subjected to extraction in order to remove all water soluble compounds. A suspension containing 5% (w/v) orange peels in deionized water was placed in a water bath (50 C) for 45 min. The suspension was filtered through a cheese cloth and the remaining solid was subjected to a second extraction applying the same conditions. Following filtration and extensive washing the remaining solid was dried at 70 C overnight. The dried material (Water Unextractable Orange Peels, WUOPs) was used throughout the present study. 2.3. Media and growth conditions WUOPs were used as solid substrate. WUOPs (2.50 g) were placed in 100 ml Erlenmeyer flasks and moistened with Toyama liquid mineral medium (Toyama et al., 1978). Consecutive optimization of enzyme production was carried out by altering, one at a time, the initial pH and the initial moisture content [initial moisture content = (weight of liquid phase/total weight of liquid and solid phase) * 100] of the medium. Depending on the microorganism, the initial culture pHs were for A. niger BTL 3.5, 5.0, 6.0 and 8.5 (Christakopoulos et al., 1990), for F. oxysporum F3 5.0, 6.0, 7.0 and 8.0 (Christakopoulos et al., 1996), for N. crassa DSM 1129, 4.0, 5.0, 5.5 and 7.0 (Macris et al., 1989) and finally for P. decumbens 5.0, 6.0, 7.0 and 8.0 (Mamma et al., 2004), at initial moisture content of 70.5% (w/w). At optimum pH, media with initial moisture contents equal to 60%, 70.5%, 80% and 90% w/w were prepared by adding the corresponding amounts of Toyama liquid mineral medium. Following heat sterilization (121 C, 20 min), the flasks were inoculated with 1 ml of spore suspension (5.8 · 107 conidia/ml) and incubated at 30 C. Samples were withdrawn periodically, extracted and assayed for enzyme activities. All experiments were performed in duplicate. 2.4. Enzyme extraction Ten volumes of distilled water were added to each flask. The extraction of the enzymes was carried out on a rotary shaker (250 rpm) at 28 ± 2 C for 1 h. The slurry was squeezed through cheese cloth. The extract was clarified by centrifugation at 12,000 · g (4 C) for 15 min. The clear supernatant was used for enzyme activity measurements. 2.5. Enzyme assays Endoglucanase, xylanase, polygalacturonase, pectate lyase and invertase activities were assayed on carboxymethyl cellulose (Sigma Chemical Co., St Louis, MO), birchwood xylan (Sigma Chemical Co., St Louis, MO), polygalacturonic acid (Sigma Chemical Co., St Louis,

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MO), pectic acid (Sigma Chemical Co., St Louis, MO) and sucrose (Sigma Chemical Co., St Louis, MO), respectively (Cheilas et al., 2000; Jayani et al., 2005). The release of reducing sugars was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). A total of 1 U of enzyme activity was defined as the amount of enzyme liberating 1 lmol of reducing sugars per min. b-Glucosidase and b-xylosidase activities were determined by a photometric assay using the respective p-nitrophenyl glycosides (Sigma Chemical Co., St Louis, MO) as substrates (Mamma et al., 1996). A total of 1 U of enzyme activity was defined as the amount of the enzyme liberating 1 lmol p-nitrophenol per min. All assays were carried out at 40 C, while the pH of the substrate was depending on the microorganism used. Enzyme activities from A. niger BTL were assayed at pH 3.5, from F. oxysporum F3 at pH 6.0, from N. crassa DSM 1129 at pH 5.0 and from P. decumbens at pH 7.0. For all enzyme assays blank samples with inactivated enzyme (after boiling for 15 min at 100 C) were used as a reference. Enzyme production was expressed in units per gram of initial dry WUOPs (U/gWUOPs). 2.6. Enzymic hydrolysis of WUOPs and production of fermentable sugars Microorganisms were grown under optimum conditions, for 6 days. Growth of microorganisms was carried out in 250 ml Erlenemeyer flasks containing 6.25 g of WUOPs. At the end of growth period 3 or 6 g of WUOPs and 92.5 or 122.5 ml, respectively, of the adequate buffer were added to the flasks containing the fermented substrate (for the buffers used see Section 2.5, all buffers contained 0.02%, w/v sodium azide). Furthermore, the fermented substrate, after the addition of 62.5 ml of the adequate buffer, was directly exposed to autohydrolysis. The flasks were incubated on a rotary shaker (250 rpm) at 49 ± 1 C. Samples were withdrawn periodically and following centrifugation (10,000 · g for 10 min), the release of reducing sugars was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). Reducing sugars were expressed, via a calibration curve, as glucose. All experiments were performed in duplicate, while measurement of reducing sugars was performed in triplicate. 2.7. Sugar analysis The hydrolysis pattern of WUOPs by the enzymic systems of the four fungi tested was obtained by HPAEC. The system was consisting of a Jasco quaternary gradient pump (Jasco PU-1580I, Jasco Ltd., UK), a Rheodyne injector and a Borwin software. The column was a CarboPack PA1, 4 · 250 mm column (Dionex Corporation, USA) with a CarboPack PA1 guard column (Dionex Corporation, USA), and the separation of sugars was monitored with a pulse amperometric detector (HPAEC-PAD) (Dionex Corporation, USA). Fucose (500 lM) was added

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in each sample as an internal standard. The column was eluted isocratically with 17.5 mM NaOH at a flow rate of 1 ml/min. The injection volume was 20 ll. All analyses were performed in triplicate. 2.8. Chemical analysis of orange peels Moisture, ash and crude fat (ether extract) content determination were conducted according to standard AOAC methods (AOAC, 1990). Pectic polysaccharides were determined using 0.25% ammonium oxalate for 4 h at 85 C (Phatak et al., 1988; Lawther et al., 1995) The cellulose, hemicellulose and (acid insoluble) lignin content were determined as described previously (AravantinosZafiris et al., 1994). Crude protein content was measured by the Kjeldhal method (Nx6.25). Chemical analysis of orange peels was performed in triplicate. 3. Results and discussion The composition of dry orange peels is presented in Table 1. Dry orange peels contained a high amount of water soluble compounds (41.1% w/w, dry basis), which were identified as glucose (14.6% w/w, dry basis), fructose (15.5% w/w, dry basis) and sucrose (10.9% w/w, dry basis), by HPAEC-PAD analysis. Orange peels are also rich in pectin, cellulose and hemicellulose (Table 1). For the purposes of the present study dry orange peels were extracted as described under Section 2 before use. It is generally agreed that the optimum medium for the enhanced production of extracellular pectinase is that containing pectic materials as an inducer (Hang and Woodanms, 1994; Solis-Pereira et al., 1993; Naidu and Panda, 1998). Furthermore, cellulases and hemicellulases production been shown to be inducible and also was affected by the nature of the substrate used in fermentation (Kang et al., 2004). Therefore, the composition of orange peels justifies its use for the production of multienzyme complexes.

Table 1 Composition of dry orange peelsa Component

% (w/w dry basis)

Crude fat Water soluble materialsb Pectin Protein Cellulose Hemicellulose Ash Lignin

3.9 ± 0.1 41.1 ± 1.2 14.4 ± 0.3 7.9 ± 0.1 16.2 ± 0.5 13.8 ± 0.3 1.7 ± 0.1 1.0 ± 0.02

Values are the mean of three determinations and the standard deviation was below 5% in all cases. a Moisture content 4.96% (w/w). b Glucose 14.6 ± 0.4% (w/w, dry basis), fructose 15.5 ± 0.5% (w/w, dry basis) and sucrose 10.9 ± 0.3% (w/w, dry basis).

3.1. Effect of initial culture pH on multienzyme production by different fungi The measurement and control of pH in SSF is very difficult. Nevertheless, the substrates employed in SSF usually have buffering effect due to their complex chemical composition. Multienzyme production by A. niger BTL, F. oxysporum F3, N. crassa DSM 1129 and P. decumbens, grown on orange peels, under SSF, initial moisture 70.5% (w/w) and different initial culture pHs was analysed over a period of 10 days. Maximum enzyme activities produced by A. niger BTL were obtained at pH 5.0 (Fig. 1a). Different initial culture pHs seems to influence more endoglucanase and xylanase production rather than the other enzymes examined. Approximately 50% less endoglucanase and xylanase activities were produced at pH 8.5 compared to optimum pH. The optimum pH for multienzyme production by F. oxysporum F3, was found to be 6.0 (Fig. 1b). Initial culture pH influence more polygalacturonase, b-glucosidase, b-xylosidase and invertase production. A decrease of 50– 65% was observed in the production of the above mentioned enzymes at pHs 5.0 and 8.0 compared to optimum pH. Increasing the initial culture pH resulted in increment of enzyme production by N. crassa DSM 1129. Maximum production was achieved at pH 5.0. A slight decrease in enzyme activities was observed at pH 7.0 compared to optimum one. b-Glucosidase production was unaffected by the initial culture pH (Fig. 1c). The fungus was unable of producing b-xylosidase activity. Initial culture pH affects strongly polygalacturonase production, by P. decumbens. Maximum polygalacturonase and pectate lyase production was achieved when the initial culture pH was 5.0. At pH 7.0 polygalacturonase production was 39% less compared to pH 5.0. Xylanase, endoglucanase and invertase production increased until the pH 7.0, while further increase of the pH caused reduction in enzyme production. No b-xylosidase activity was detected. b-Glucosidase production was practically the same at all pHs tested (Fig. 1d). Christakopoulos et al. (1999) reported that acetyl esterase production by F. oxysporum F3 was unaffected by the initial pH of the medium when pH values of 5, 6, or 7 were examined. 3.2. Effect of initial moisture content on multienzyme production by different fungi Moisture content is a critical factor on SSF processes because this variable has influence on growth and biosynthesis and secretion of different metabolites (Krishna and Chandrasekaran, 1996; Ellaiah et al., 2002). Lower moisture content causes reduction in solubility of nutrients of the substrate, low degree of swelling and high water tension. On the other hand, higher moisture levels can cause a reduction in enzyme yield due to steric hindrance of the

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Fig. 1. Effect of initial culture pH on the maximum level of polygalacturonase (PGA), pectate lyase (PL), endoglucanase (EG), xylanase (XYL), b-glucosidase (b-GLU), b-xylosidase (b-XYL) and invertase (INV) produced by (a) A. niger BTL, Symbols: (j) pH 3.5, (h) pH 5.0, ( ) pH 6.0, ( ) pH 8.5. (b) F. oxysporum F3, Symbols: (j) pH 5.0, (h) pH 6.0, ( ) pH 7.0, ( ) pH 8.5, (c) N. crassa DSM 1129, Symbols: (j) pH 4.0, (h) pH 5.0, ( ) pH 5.5, ( ) pH 7.0 and (d) P. decumbens, Symbols: (j) pH 5.0, (h) pH 6.5, ( ) pH 7.0, ( ) pH 8.0, grown, under solid-state fermentation, on WUOPs as sole carbon source (initial moisture content: 70.5%, w/w).

growth of the producer strain by reduction in porosity (interparticle spaces) of the solid matrix, thus interfering oxygen transfer (Lonsane et al., 1985). As the optimal value of moisture content depends on both the microorganism and the solid matrix used, for economical production, the microorganism should be grown in optimal moisture levels either for maximising the growth or metabolite production (enzymes, organic acids, etc.) depending on the application. The four fungal strains of the present study were grown at optimum pH and at four different initial moisture contents, namely 60%, 70.5%, 80% and 90% (w/w). Enzyme production was monitored over a period of 10 days. Enzyme production by A. niger BTL was profoundly affected by initial moisture content of the culture. Increas-

ing moisture content resulted in considerable increase in enzyme production. Increment in enzyme production is depending on the particular enzyme and ranged from 2.5 times to 9 times. For example, increasing moisture content from 60% (w/w) to 90% (w/w) pectate lyase production increased approximately 9 times, while xylanase only 2.5 times (Table 2). Consequently, optimum moisture content for A. niger BTL was found to be 90% (w/w). The effect of initial moisture content on multienzyme production by F. oxysporum F3 followed the same trend as for A. niger BTL. Increasing moisture content from 60% (w/w) to 90% (w/w) resulted in 3.5–17 times increase in enzyme production, depending on the particular enzyme (Table 2). Pectate lyase production, was the most affected by initial moisture content. Optimum initial moisture

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Table 2 Effect of initial moisture content (%, w/w) on the maximum level of polygalacturonase, pectate lyase, endoglucanase, xylanase, b-glucosidase, b-xylosidase and invertase (Units/gWUOPs) produced by A. niger BTL (initial culture pH 5.0) and F. oxysporum F3 (initial culture pH 6.0) grown, under solid-state fermentation, on WUOPs as sole carbon source Initial moisture (%, w/w)

Polygalacturonase

Pectate lyase

Endoglucanase

Xylanase

b-Glucosidase

b-Xylosidase

Invertase

A. niger BTL 60 70.5 80 90

29.4 ± 0.9 48.6 ± 1.7 89.0 ± 2.7 135.7 ± 4.1

15.1 ± 0.4 48.9 ± 1.5 87.8 ± 2.1 130.8 ± 3.9

9.2 ± 0.2 12.9 ± 0.4 25.0 ± 0.7 60.5 ± 1.8

30.3 ± 0.9 54.9 ± 1.6 52.0 ± 1.5 77.1 ± 2.3

0.80 ± 0.02 1.80 ± 0.04 2.18 ± 0.07 3.14 ± 0.09

0.21 ± 0.01 0.38 ± 0.01 0.50 ± 0.02 1.04 ± 0.03

18.3 ± 0.5 32.1 ± 0.6 51.5 ± 1.6 72.5 ± 2.2

17.1 ± 0.4 33.0 ± 1.0 46.8 ± 1.5 91.4 ± 2.7

2.1 ± 0.1 8.0 ± 0.2 19.9 ± 0.6 38.0 ± 1.1

14.2 ± 0.3 37.8 ± 0.9 46.0 ± 1.4 69.5 ± 2.1

3.1 ± 0.1 5.2 ± 0.2 16.8 ± 0.3 28.9 ± 0.9

0.32 ± 0.01 0.55 ± 0.02 0.68 ± 0.02 0.92 ± 0.03

0.007 ± 0.0 0.007 ± 0.0 0.065 ± 0.001 0.092 ± 0.003

6.9 ± 0.2 19.9 ± 0.5 20.8 ± 0.6 24.6 ± 0.7

F. oxysporum F3 60 70.5 80 90

Values are the mean of four determinations and the standard deviation was below 4% in all cases.

content for multienzyme production by F. oxysporum F3 was found to be 90% (w/w). Production of different enzymes by N. crassa DSM 1129 increased 1.5–3.5 times when the initial moisture content of the culture increased from 60% to 80% (w/w). Enzyme activities except for invertase, reached their maximum values at 80% (w/w) moisture. Reduction in enzyme production except for invertase, was observed at initial moisture equal to 90% (w/w) (Table 3). Increasing initial moisture from 60% (w/w) to 90% (w/w) resulted in 2–7.5 times increment in enzyme production by P. decumbens. All enzymes reached their maximum values at 90% (w/w) initial moisture (Table 3). A. niger BTL was by far the most potent strain in polygalacturonase and pectate lyase production, followed by F. oxysporum F3. This result was expected since the fungal genera Aspergillus, Rhizopus and Trichoderma are the chief sources of pectinolytic enzymes (Blandino et al., 2001). As far as endoglucanase and b-glucosidase production is concerned N. crassa DSM 1129 produced the highest activities. Comparison of xylanase production revealed that A. niger BTL produced the highest activity, followed by N. crassa DSM 1129. Finally, the highest invertase activity was pro-

duced by A. niger BTL. No b-xylosidase activity was detected in culture extracts of N. crassa DSM 1129 and P. decumbens, while A. niger BTL produced approximately 10 times more b-xylosidase than F. oxysporum F3. The quantities of polygalacturonase obtained in the present study by all four fungal strains were high compared to those reported for pectinolytic strains such as A. niger (25 U/gsubstrate), P. italicum (25 U/gsubstrate), P. frequentans (3.4 U/gsubstrate) cultivated on solid substrates (Castilho et al., 1999, 2000; Hours et al., 1988; Garzo´n and Hours, 1992). The thermophilic fungus Thermoascus aurantiacus, when grown in a media containing wheat bran or orange bagasse produced 43 U/gsubstrate of polygalacturonase (Martins et al., 2002). Furthermore, a yield of 35 units of exo-pectinase per gram dry substrate has been achieved in SSF using a strain of A. niger (Solis-Pereira et al., 1996). Citrus waste has been used mainly for pectinase production under submerged culture by different fungi. High levels of endo-polygalacturonase were produced by Aureobasidiurn pullulans on orange-peel waste (Federici and Petruccioli, 1985) and by Aspergillus sp. using untreated lemon peel as a carbohydrate source (Larios et al., 1989). Further-

Table 3 Effect of initial moisture content (%, w/w) on the maximum level of polygalacturonase, pectate lyase, endoglucanase, xylanase, b-glucosidase and invertase (Units/gWUOPs) produced by N. crassa DSM 1129 (initial culture pH 5.0) and P. decumbens (initial culture pH 7.0) grown, under solid-state fermentation, on WUOPs as sole carbon source Initial moisture (%, w/w)

Polygalacturonase

Pectate lyase

Endoglucanase

Xylanase

b-Glucosidase

Invertase

N. crassa DSM 1129 60 70.5 80 90

26.7 ± 0.7 56.7 ± 1.7 63.3 ± 1.9 58.3 ± 1.6

12.8 ± 0.4 28.4 ± 0.9 29.1 ± 0.9 26.2 ± 0.8

84.7 ± 2.9 108.9 ± 2.2 138.5 ± 4.2 40.7 ± 1.0

40.9 ± 1.2 49.5 ± 1.5 56.8 ± 1.7 37.4 ± 1.1

2.3 ± 0.1 4.6 ± 0.1 7.9 ± 0.2 1.9 ± 0.1

24.5 ± 0.6 44.0 ± 1.5 65.0 ± 1.9 74.0 ± 1.8

P. decumbens 60 70.5 80 90

17.1 ± 0.5 40.9 ± 1.2 48.1 ± 1.2 56.3 ± 1.7

13.7 ± 0.4 36.4 ± 1.1 43.3 ± 1.3 49.6 ± 1.5

20.8 ± 0.6 26.6 ± 0.7 42.7 ± 1.3 45.5 ± 1.4

5.1 ± 0.2 15.7 ± 0.5 17.9 ± 0.4 37.8 ± 1.1

0.42 ± 0.01 0.68 ± 0.02 1.23 ± 0.04 3.19 ± 0.10

13.6 ± 0.3 21.3 ± 0.6 25.4 ± 0.9 24.7 ± 0.7

Values are the mean of four determinations and the standard deviation was below 4% in all cases.

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more, orange pulp pellets and dried citrus peel were used for pectinase production by Tubercularia vulgaris and A. niger, respectively under submerged culture (Fonseca and Said, 1994; Dhillon et al., 2004), while alkali-treated orange peels were used for xylanase production under submerged culture by Streptomyces actuosus (Wang et al., 2003). Ismail, 1996) reported the production of pectinases, cellulases and xylanases using six fungal isolates, of the genera Aspergillus, Penicillium and Memmoniella, grown on orange peels as sole carbon source. The comparison of pectinase levels produced by fungi is not straightforward, because distinct culture conditions have been used. In addition, the composition of pectinase complex is different for each fungus and, finally because several methods for enzyme activity have been used (Fonseca and Said, 1994). Endoglucanase activity obtained in the present study by all fungal strains examined is higher than that produced by A. ustus, grown on rice straw or wheat bran (12.6 and 11.8 U/gsubstrate, respectively) (Shamala and Sreekantiah, 1986). A. niger KK2 grown on rice straw produced comparable endoglucanase activity (130 U/gsubstrate) (Kang et al., 2004), to N. crassa DSM 1129. P. capsulatum, Trichoderma reesei MCG77, Talaromyces emersonii UCG208 and T. aurantiacus grown on beet pulp, wheat bran or wheat straw produced much higher endoglucanase, b-glucosidase, xylanase and b-xylosidase activities than those reported in the present study (Considine et al., 1988; Tuohy et al., 1990; Kalogeris et al., 1999). Usually production of cellulases and hemicellulase is carried out on lignocellulosic materials. Enzyme production is strongly affected by substrate composition. For example, F. oxysporum F3 grown on corn stover under solid-state fermentation produced endoglucanase, b-glucosidase, xylanase, b-xylosidase activities of 211, 0.088, 1216, 0.052 U/gsubstrate, respectively (Panagiotou et al., 2003). These values are much higher than those reported in the present study, by the same microorganism grown on different carbon source. The results indicate that orange peels is suitable for multienzyme production by the fungal strains tested and that SSF may be achieved using this agro-industrial residue. 3.3. Hydrolysis of WUOPs by the enzymic systems of the four fungi and production of fermentable sugars Topakas et al. (2004), have successfully applied a twostage process which included SSF growth of Sporotrichum thermophile on corn cobs followed by the autohydrolysis of the fermented substrate and the liberation of hydroxycinnamic acids. The same approach was applied in the case of WUOPs. The four fungi tested, were grown under optimal conditions on WUOPs. The hydrolysis of the fermented WUOPs or the added amounts of it, by the extracellularly produced enzymes was monitored. Release of reducing sugars from fermented as well as from the added amounts of WUOPs by the enzymic systems of the four fungi tested is presented on Fig. 2.

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Reducing sugars produced by autohydrolysis of fermented WUOPs by the enzymic system of A. niger BTL reached 20.1 g/l (yield 249.7 mg/gtotal WUOPs), while the values for 3 and 6 g addition of WUOPs were 29.6 (yield 344.4 mg/gtotal WUOPs) and 24.1 g/l (yield 270.4 mg/ gtotal WUOPs) respectively at the end of hydrolysis (168 h). In all cases reducing sugar release increased progressively up to 120 h and remained constant after that time (Fig. 2a). The HPAEC-PAD analysis revealed that the hydrolysis of WUOPs in all cases resulted in monosaccharides, mainly in pentose (xylose + arabinose) and less in hexose production (glucose + galactose). During autohydrolysis of the fermented WUOPs pentose fraction was 3.5 times higher than hexose fraction. Xylose was the major pentose product, while equal amounts of glucose and galactose were found (Fig. 3a). The addition of 3 g WUOPs in the fermented material resulted in 27% and 48% incerement of the pentose and hexose fractions respectively. Xylose concentration remained practically constant, compared to autohydrolysis. Arabinose and galactose concentrations were increased significantly compared to the respected values during autohydrolysis, while glucose increased slightly (Fig. 3a). Finally, the addition of 6 g WUOPs resulted in decrease in reducing sugar release with concomitant decrease in monosaccharides concentration (Figs. 2a and 3a). Decrease in sugar release could be attributed to inhibition of enzyme action. Autohydrolysis of fermented WUOPs by the enzymic system of F. oxysporum F3 resulted in 10.90 g/l reducing sugars (yield 132.28 mg/gtotal WUOPs), while 3 and 6 g addition in 9.25 (yield 107.50 mg/gtotal WUOPs) and 8.07 g/l (yield 90.58 mg/gtotal WUOPs) respectively at the end of hydrolysis time (168 h) (Fig. 2b). Release of reducing sugars increased progressively up to 120 h, while until 168 h a slight increase was observed. The addition of different amounts of WUOPs seems to influence enzyme action, since increment in the added amount of WUOPs resulted in decrease in reducing sugar concentration (Fig. 2b). In all cases the main sugars detected by HPAEC-PAD analysis were arabinose, xylose, glucose and the disaccharide xylobiose. In contrast to A. niger BTL, glucose was the major product obtained by the enzymatic system of F. oxysporum F3. Glucose concentration approximately doubled when 3 g of WUOPs were added, but decreased by the addition of 6 g WUOPs. The pentose fraction (arabinose + xylose) increased when additions of WUOPs were made. Xylobiose was accumulated in all cases and that could be attributed to low b-xylosidase activity produced by F. oxysporum F3 when grown on WUOPs (Fig. 3b). Reducing sugar release during the autohydrolysis of fermented WUOPs by the enzymic system of the fungi N. crassa DSM 1129 reached 14.79 g/l (yield 222.45 mg/ gtotal WUOPs), while the values for 3 and 6 g addition of WUOPs were 22.78 (yield 264.79 mg/gtotal WUOPs) and 18.32 g/l (yield 205.67 mg/gtotal WUOPs) respectively at the end of hydrolysis (182 h) (Fig. 2c). In all cases the main sugars detected by HPAEC were arabinose, xylose, glucose,

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Fig. 2. Time course of reducing sugars release during the hydrolysis of WUOPs by the enzymic systems of (a) A. niger BTL, (b) F. oxysporum F3, (c) N. crassa DSM 1129 and (d) P. decumbens. Symbols: (d) autohydrolysis, (s) hydrolysis of 3 g added WUOPs (.) hydrolysis of 6 g added WUOPs.

galactose and the disaccharide xylobiose (Fig. 3c). Autohydrolysis of fermented WUOPs resulted in an hexose fraction (glucose + galactose) to pentose (arabinose + xylose) ratio approximately 2:1, while xylobiose reached 1.35 g/l, approximately four times higher concentration than that observed when the same conditions applied to F. oxysporum F3. Addition of 3 g WUOPs resulted in increased glucose and xylose concentrations while arabinose and galactose concentrations were decreased, compared to autohydrolysis. Further decrease in arabinose and galactose concentrations was detected after addition of 6 g WUOPs. In this case a decrease in glucose concentration was also observed, while xylose concentration remained practically constant. Xylobiose continues to accumulate after 3 and 6 g addition of WUOPs (Fig. 3c). As mentioned earlier N. crassa DSM 1129 was unable of producing b-xylosidase activity which is the reason for xylobiose accumulation. Reducing sugars produced by autohydrolysis of fermented WUOPs by the enzymic system of P. decumbensreached 9.22 g/l (yield 114.32 mg/gtotal WUOPs), while the values for 3 and 6 g addition of WUOPs were 15.96

(yield 185.50 mg/gtotal WUOPs) and 22.76 g/l (yield 255.51 mg/gtotal WUOPs) respectively at the end of hydrolysis (144 h). Reducing sugars release during autohydrolysis as well as hydrolysis after the addition of 3 g WUOPs increased progressively up to 72 h and remained constant after that time, while in the case of 6 g addition increment of reducing sugars release was observed up to 120 h (Fig. 2d). The HPAEC-PAD analysis revealed that the hydrolysis products in all cases were glucose, galactose, xylose and arabinose. Glucose was the major hexose product which increased when additions of 3 or 6 g WUOPs were made (Fig. 3d). Galactose concentration increased in 3 g addition and decreased with further addition of WUOPs. The same pattern was observed with the pentose fraction, where xylose concentration increased after the additions of WUOPs, while arabinose concentration increased after the 3 g addition and decreased with further addition (Fig. 3d). It should be noted that even though, P. decumbens when grown on WUOPs was unable of producing b-xylosidase activity, no xylobiose was detected by HPAEC-PAD analysis.

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Fig. 3. Mono- and di-saccharides produced during hydrolysis of WUOPs by the in situ produced multienzyme systems of (a) A. niger BTL, (b) F. oxysporum F3, (c) N. crassa DSM 1129 and (d) P. decumbens. Symbols: (h) arabinose, (j) xylose, ( ) glucose, ( ) galactose and ( ) xylobiose.

Differences in hydrolysis products of WUOPs by the enzymic systems of the four fungi tested could be attributed to different enzyme activities when grown on the above mentioned substrate. Furthermore, the total concentration of the sugars detected by HPAEC-PAD analysis counts a percentage ranged from 40% to 80%, depending on the fungi and condition, of the reducing sugars measured by DNS method. This difference could probably be attributed to uronic acids produced during hydrolysis of substrate’s the pectin content. The results indicate that enzymic conversion of polymeric carbohydrates in orange peels can provide a promising route for the production of monomeric sugars for further conversion to value-added products. 4. Conclusions In conclusion, it was possible to control simultaneous production of pectinolytic, cellulolytic and xylanolytic enzymes by fungal strains of the genera Aspergillus, Fusar-

ium, Neurospora and Penicillium and generate multienzyme activities using a simple growth medium consisting of a solid by-product of the citrus processing industry (orange peels) and a mineral medium. Furthermore, the two-stage process proposed which includes coupling enzymic treatment and solid-state fermentation, resulted in the production of fermentable sugars which could be converted to bioethanol. These findings make this process worthy of further investigation. References Acun˜a-Argu¨elles, M.E., Gutie´rrez-Rojas, M., Viniegra-Gonza´les, G., Favela-Torres, E., 1994. Effect of water activity on exo-pectinase production by Aspergillus niger CH4 on solid state fermentation. Biotechnol. Lett. 16, 23–28. Antier, P., Minjares, A., Roussos, S., Raimbault, M., Viniegra, G., 1993. Pectinase-hyperproducing mutants of Aspergillus niger C28B25 for solid state fermentation of coffee pulp. Enzyme Microb. Technol. 15, 254–260. AOAC, 1990. Official Methods of Analysis of the Association of Official Analytical Chemists, 15th ed. AOAC Inc., USA.

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